A family of loci homologous to the EGF-like portion of Notch, a gene involved in neurogenesis, have been identified in D. melanogaster. The sequence, spatial, and temporal distribution of both RNA and protein of one of these loci suggest a possible role in the development of the central nervous system (CNS). In situ hybridization and antibody staining of embryos show initial localization in cells along the midline of the neuroepithelium. High level expression is restricted in the developing embryo to a subset of six midline glial cells abutting growing axons. Extracellular localization is suggested by the presence of EGF-like repeats in the deduced protein sequence and antibody staining. Cytological, immunocytochemical, genetic, and molecular data show that this gene corresponds to the slit locus. Mutations in this locus result in the collapse of the regular scaffold of commissural and longitudinal axon tracts in the embryonic central nervous system.